N-Hydroxyacetamine has been proposed to be the initial step in the formation of a reactive metabolite of acetaminophen. Once formed this compound was postulated to spontaneously dehydrate to the reactive compound N-acetamyl-imidoquinone. This substrate was recently synthesized by Dr. I.C. Calder in 0.05 M Tris buffer, ph 7.4, with a half-life of 15 min. We obtained a sample of the compound as a gift to study the mechanism of its conjugation GSH. In the presence of glutathione (GSH) and H2180, the compound formed a GSH- acetaminophen conjugate with no incorporation of 180. In other work we showed in conversion of p-180-acetamino to a reactive metabolite there was no loss of 180. The half-life for conversion of N-hydroxyacetaminophen to a reactive metabolite in the presence of GSH, however was 1 hr. In the presence of microsomes and GSH, the half-life was also 1 hr..